This application relates to high pressure liquid chromatography (HPLC) and to chromatographic columns employed therein. More particularly, this invention relates to chromatographic columns which may be modularly modified as to length and/or internal diameter and to components employed therewith. This invention also relates to uses thereof.
Chromatography is a separation method whereby individual chemical compounds which were originally present in a mixture are resolved from each other by the selective process of distribution between two heterogenous (immiscible) phases. The distribution of chemical species to be separated occurs in a dynamic process between the mobile phase and the stationary phase. The stationary phase, or the column packing material, usually has a relatively large surface area, through which the mobile phase is allowed to flow. The chemical nature of the stationary phase exercises the primary control over the separation process. The greater the affinity of a particular chemical compound to the stationary phase, the longer it will be maintained in the system. The mobile phase can be either gas or liquid; correspondingly, the methods are referred to as gas chromatography and liquid chromatography.
There are four combinations of heterogenous phase systems, which give rise to four different chromatographic methods: gas-solid, liquid-solid, gas-liquid, and liquid-liquid chromatography. In gas-solid and liquid-solid chromatography, sample molecules are caused to interact physically with the surface of a porous solid by means of a phenomenon called adsorption. Hence, these two methods are also generally referred to as adsorption chromatography. The adsorption effect of the chromatographic column packing material determines their rates of migration through the column. In gas-liquid and liquid-liquid chromatography, the liquid stationary phase is held on the surface of an inert solid which serves merely as its support and, ideally, does not participate in the separation process. Primarily, then, the components of a mixture of chemicals having different solubilities in the stationary phase separate by migrating at different rates. The partitioning of the mixture between the two phases is the basis of these methods, they are also generally referred to as partition chromatography. The rate of migration of the various components of the mixture can be related to its thermodynamic partition coefficient in a given two-phase system.
Chromatography has been used primarily as a separation and isolation method. Unlike classical chemical separation methods (for example, precipitation or crystalization), chromatography is intended to separate many mixture components in a single-step procedure. Chromatographic methods can be applied to an incredible concentration range: although some separations can be performed in commercial-scale quantitites (e.g., milligrams to grams, grams to kilograms) which we refer to as preparative HPLC, and it is also feasible to carry out certain highly sensitive analytical determinations on the order of 10.sup.-9 g Chromatographic separations are based on the physiochemical principles of adsorption and partition, and, conversely, these and related fundamental phenomena can be studied by highprecision chromatography of model systems.
In liquid chromatography, the mobile phase may be percolated through the column at atmospheric pressure, by means of gravity, or under more contemporary precedures through pressure generated by a suitable pump. High pressure pumps which can generate up to several thousand atmospheres of inlet pressure are used.
Thus, high pressure liquid chromatography is a process of separating complex mixtures of chemicals by passing a solution of the chemicals through a stationary column packing material and under a certain degree of pressure. Preparative high pressure liquid chromatography is that process where one applies a mixture of a compound in a solution in its mobile phase where the selectivity of the components of the column packing material will retain the various and select components of the mixture for that length of time which causes its select components to be eluted discretely for separate collection as individual components and as purified elements. The efficiency of the columns is what makes such separation possible, and the efficiency of the columns depend upon such factors as the following:
(1) Length and/or width of the column.
(2) Type of medium.
(3) The mobile phase.
(4) Complex to be separated.
The eluted components are collected as fractions of the analyzed or prepared chemical mixture.
This invention, more specifically, relates to the configuration and structure of the columns used in high pressure liquid chromatography, and particularly in preparative chromatography. Through usage of the mechanisms of this particular invention the chromatographer can optimize preparative HPLC by permitting the best possible combinations of column internal diameters (ID) and lengths. This is readily accomplished by coupling columns of varying lengths through the use of the variety of adaptors employed in the build up of columns for use in the method of this invention. The system of this invention assembles quite easily mainly because all of its components are totally modular, and they are very convenient to handle.
With regard to the usage of this particular invention for preparative high pressure liquid chromatography, for performance of semi-preparative HPLC, which is used for separating miligram to gram quantities, the columns will normally be subjected to pressures up to approximately 2,000 psi. And, the type of columns suggested for usage are those which will have internal diameters of between about 1/2 inch to 1 inch. Usage of this current invention for preparative HPLC, such as for heavier loadings of grams to 10 grams quantities, the type of columns recommended would be those within a range of having an internal diameter of between about 1 inch to 4 inches in dimension, and which also functions up to approximately 2,000 psi of pressure. In process liquid chromatography, which includes the separations of grams to kilogram quantities, it is recommended that the modular columns of this invention incorporate internal diameters of between about 4 inches, or through columns having diameters approximating 5 inch or 6 inch, or even above. And, the pressure rating for these type of columns generally is within the range of 150 psi of pressure. Column sections obtainable for usage in conjunction with the teachings of this invention are available in a variety of lengths, as from 5 centimeters, or less, up to 100 centimeters, or more, and may include diameters up to approximately 20 inches. Thus, it can be readily seen that the flexibility that is built into the modularization or combination of chromatographic columns in conjunction with this invention add significant flexibility to the processes that can be undertaken by the lab technician, and by using the variety of additional adaptor components to be subsequently analyzed, such as a cone adaptor, or regular adaptor, including end plates, a variety of chromatographic columns having differing internal diameters can be achieved, and function quite effectively to attain the desired results. Another unique feature of this type of invention is that it may be used when heavier loadings of the chromatographic columns are desired.
Lengthwise, the various preparative columns, whether it be of the semi-preparative type, for use in preparative HPLC, or for process HPLC, normally such columns are available in lengths of approximately 5 cm up to 100 cm in length. Although, HPLC, normally column lengths, of the 4 inch type, usually begin with a minimum length of approximately 20 cm. The semipreparative column is considered to be a column with an internal diameter, as previously explained, of about a 1/2 in. or 1 in., and more generally is in the 30 or 50 cm of length category, packed with particles of between about 10 microns to 40-60 microns. Although, this current invention has been found to function quite effectively even with shorter length columns, particularly since the various columns of this particular invention are of the modular type, and can be built up one upon the other to provide the desired length for the overall column that may be deemed necessary to attain effective and efficient chromatography. Obviously it depends upon the mobile phase being subjected to chromatography, and the type of packing provided within the column that controls and determines the length of column necessary, and whether or not even shorter columns may be employed.